We will test novel targeted strategies to modulate the emergence and evolution of drug resistance using the NEDD8-activating enzyme (NAE) inhibitor MLN4924. Aim 1 focuses on evaluating how treatment-associated on-target mutations influence regression and relapse in spontaneous disease models. Ex-vivo strategies designed to control the types and frequencies of known MLN4924-resistance mechanisms will be compared to in vivo treatment-associated relapse to delineate how selective pressure by a strongly mono-targeted drug drives cancer cell evolution. Aim 2 tests a strategy that uses a less selective NAE inhibitor to transiently suppress MLN4924 resistance by providing a low level, secondary selective pressure. Aim 3 determines how providing distinct selective pressures at a known drug resistance hotspot in NAE influence the type and frequency of MLN4924 treatment-emergent resistance mechanisms. Collectively, our studies using the clinical NAE inhibitor MLN4924 will establish new rational paradigms for targeted therapies designed to suppress and potentially reverse treatment-emergent drug resistance.